Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating enzymes Ubc6 and Ubc7 confer resistance to hygromycin B in Saccharomyces cerevisiae

Aberrant endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed through ER-associated degradation (ERAD) and INM-associated degradation (INMAD). We previously showed the Hrd1, Doa10, and Asi ERAD and INMAD ubiquitin ligases (E3s) in Saccharomyces cerevisiae confer resistance to hygromycin B, which distorts the ribosome decoding center. Here, we assessed the requirement of Ubc6 and Ubc7, the primary ERAD and INMAD ubiquitin-conjugating enzymes (E2s) for hygromycin B resistance. Loss of either E2 sensitized cells to hygromycin B, with UBC7 deletion having a greater impact, consistent with characterized roles for Ubc6 and Ubc7 in ER and INM protein quality control.


Description
Degradation of misfolded, excess, and otherwise aberrant proteins is critical for cellular homeostasis.The ability to recognize and destroy faulty proteins declines with age, and disruptions to enzymes contributing to protein quality control (PQC) contribute to several diseases (Badawi et al., 2023;Guerriero & Brodsky, 2012).Eukaryotic cells possess compartmentspecific PQC mechanisms, including those dedicated to the turnover of aberrant proteins at the physically continuous endoplasmic reticulum (ER) membrane and inner nuclear membrane (INM) (Mehrtash & Hochstrasser, 2019).ER-associated degradation (ERAD) promotes turnover of aberrant ER luminal, transmembrane, and translocon-clogging proteins as well as cytosolic polypeptides that contact the ER surface.INM-associated degradation (INMAD) mediates proteolysis of faulty INM transmembrane and INM-abutting soluble nucleoplasmic proteins.ERAD and INMAD both employ ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) to polyubiquitylate proteins (Figure 1A), destining them for destruction by cytosolic or nucleoplasmic proteasomes.
To assess the roles of Ubc6 and Ubc7 in combatting proteotoxic stress caused by hygromycin B, we cultured serial dilutions of wild type yeast, yeast lacking UBC6 and UBC7 individually or in concert, as well as a yeast strain rendered broadly defective for ERAD and INMAD by simultaneous deletion of HRD1, DOA10, and ASI1 (Figure 1B).All strains grew similarly in the absence of hygromycin B. Loss of either UBC6 or UBC7 sensitized yeast to hygromycin B, with UBC7 deletion having a stronger impact.Combined deletion of both UBC6 and UBC7 caused a greater growth defect than individual absence of either E2-encoding gene.Finally, hrd1Δ doa10Δ asi1Δ yeast exhibited a more profound growth defect than any E2 mutant.
To validate these results, we assessed hygromycin B sensitivity of ubc6Δ and ubc7Δ yeast strains in a distinct genetic background, as well as three double mutants lacking catalytic components of the ERAD or INMAD E3s (Figure 1C).As before, loss of either E2 sensitized yeast to hygromycin B, with cells lacking Ubc7 faring more poorly than those without Ubc6.Loss of any two ERAD or INMAD E3s approximately phenocopied ubc7Δ yeast.
A greater role for Ubc7 than Ubc6 in combatting proteotoxicity likely reflects broader Ubc7 participation in ERAD and INMAD.Loss of Ubc6 is expected to compromise Doa10 and Asi function, while UBC7 deletion is predicted to abolish all three major branches of ERAD and INMAD.The observation that ubc6Δ ubc7Δ double mutant yeast exhibit a stronger growth defect than either ubc6Δ or ubc7Δ single mutant suggests independent functions for both Ubc6 and Ubc7.Identification of Ubc6-dependent, Ubc7-independent PQC substrates would support this model.Further, an enhanced growth defect of hrd1Δ doa10Δ asi1Δ compared to ubc6Δ ubc7Δ yeast is in agreement with other reports indicating additional E2s (such as Ubc1) may function with ERAD and INMAD E3s, when the primary E2s are unavailable.
Our data are consistent with a previous study demonstrating overexpression of genes encoding either E2 enhances resistence to multiple stresses, including heat stress, oxidative stress, and presence of the toxic amino acid analog canavanine (Hiraishi et al., 2006).Conversely, previous work showed that ubc7Δ and hrd1Δ doa10Δ yeast exhibited similar hypersensitivity to cadmium (Swanson et al., 2001).Large-scale analyses indicated loss of UBC7 reduces tolerance to multiple transition metals, which oxidatively damage a range of biological macromolecules, including proteins (Bleackley et al., 2011;Ruotolo et al., 2008;Zhao et al., 2020), and genotoxic agents (Alamgir et al., 2010;Brown et al., 2006;Gaytan et al., 2013;Kapitzky et al., 2010).We note hygromycin B hypersensitivity was not observed for ubc6Δ or ubc7Δ yeast in a previous report (Chuang & Madura, 2005).This may be due to differences in effective drug concentrations in culture medium.In alignment with our results, we have also recently shown that loss of Doa10, Hrd1, and Ubc7 homologs sensitizes the pathogenic fungi Candida albicans to hygromycin B (Doss et al., 2023).Overall, our work supports a critical and conserved function for endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating enzymes in protein quality control.

Growth assays
Yeast growth was analyzed as previously described (Watts et al., 2015).Briefly, sixfold dilutions of each yeast strain were spotted onto yeast extract-peptone-dextrose medium (Guthrie & Fink, 2004) lacking or containing hygromycin B (Gibco) at the indicated concentrations and incubated at 30°C for the indicated amount of time.

Figure 1 .
Figure 1.UBC6 and UBC7 confer resistance to hygromycin B: (A) Endoplasmic Reticulum (ER)-Associated Degradation and Inner Nuclear Membrane (INM)-Associated Degradation pathways.In conjunction with the E2 Ubc7, the E3 Hrd1 promotes degradation of aberrant ER luminal and transmembrane proteins as well as proteins that clog ER translocons.The E3 Doa10 functions with two E2s, Ubc6 and Ubc7, to mediate degradation of aberrant transmembrane proteins at the ER or INM in addition to soluble cytosolic or nucleoplasmic proteins.The trimeric Asi E3 complex (Asi1, Asi2, and Asi3) works with Ubc6 and Ubc7 to target aberrant transmembrane INM and soluble nucleoplasmic proteins.Ubc7 is anchored at the ER membrane through interaction with Cue1.Ub, ubiquitin.(B) and (C) Sixfold serial dilutions of yeast of the indicated genotype were spotted on medium lacking (No Drug) or containing increasing concentrations of hygromycin B. Plates were incubated at 30°C and imaged after 1-2 days.Experiments were performed three or more times.